Using in vivo-biotinylated ubiquitin to describe a mitotic exit ubiquitome from human cells

Mitotic division requires highly regulated morphological and biochemical changes to the cell. Upon commitment to exit mitosis, cells begin to remove mitotic regulators in a temporally and spatially controlled manner to bring about the changes that re-establish interphase. Ubiquitin-dependent pathways target these regulators to generate polyubiquitin-tagged substrates for degradation by the 26S proteasome. However, the lack of cell-based assays to investigate in vivo ubiquitination limits our knowledge of the identity of substrates of ubiquitin-mediated regulation in mitosis. Here we report an in vivo ubiquitin tagging system in human cells that allows efficient purification of ubiquitin conjugates from synchronised cell populations. Coupling purification with mass spectrometry, we identify a series of mitotic regulators that are targeted for polyubiquitination in mitotic exit. We show that some are new substrates of the Anaphase Promoting Complex/Cyclosome (APC/C), and validate KIFC1 and RacGAP1/Cyk4 as two such targets involved respectively in timely mitotic spindle disassembly and cell spreading. We conclude that in vivo biotin-tagging of ubiquitin can provide valuable information about the role of ubiquitin-mediated regulation in processes required for rebuilding interphase cells

Long-pulse laser launch and ionization of tailored large neutral silver nanoparticles with atomic mass assignment

We explore the synthesis, characterization, neutral launch and vacuum ultraviolet ionization of massive perfluorinated-alkyl-capped nanoparticles. The presence of the ligand coating in solution is corroborated by Fourier transform infrared spectroscopy (FT-IR) and the particle size distribution is analyzed by transmission electron microscopy (TEM). Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry identifies perfluoralkyl coated silver nanoparticles as the most stable species among the materials studied here. They can be launched in high vacuum using long-pulse low-power laser heating-orders of magnitude below typical thresholds for laser desorption. Energy-dispersive X-ray spectroscopy (EDX) of the recaptured silver clusters confirms the expected elemental distribution. Volatilization with subsequent ionization of the neutral nanoparticle beam in high vacuum by 157 nm light allows analyzing their mass with atomic resolution

Quantitative proteomic analysis of Parkin substrates in Drosophila neurons.

Parkin (PARK2) is an E3 ubiquitin ligase that is commonly mutated in Familial Parkinson's Disease (PD). In cell culture models, Parkin is recruited to acutely depolarised mitochondria by PINK1. PINK1 activates Parkin activity leading to ubiquitination of multiple proteins, which in turn promotes clearance of mitochondria by mitophagy. Many substrates have been identified using cell culture models in combination with depolarising drugs or proteasome inhibitors, but not in more physiological settings.Here we utilized the recently introduced BioUb strategy to isolate ubiquitinated proteins in flies. Following Parkin Wild-Type (WT) and Parkin Ligase dead (LD) expression we analysed by mass spectrometry and stringent bioinformatics analysis those proteins differentially ubiquitinated to provide the first survey of steady state Parkin substrates using an in vivo model. We further used an in vivo ubiquitination assay to validate one of those substrates in SH-SY5Y cells.We identified 35 proteins that are more prominently ubiquitinated following Parkin over-expression. These include several mitochondrial proteins and a number of endosomal trafficking regulators such as v-ATPase sub-units, Syx5/STX5, ALiX/PDCD6IP and Vps4. We also identified the retromer component, Vps35, another PD-associated gene that has recently been shown to interact genetically with parkin. Importantly, we validated Parkin-dependent ubiquitination of VPS35 in human neuroblastoma cells.Collectively our results provide new leads to the possible physiological functions of Parkin activity that are not overtly biased by acute mitochondrial depolarisation

Vibrational spectrum of topologically disordered systems

The topological nature of the disorder of glasses and supercooled liquids strongly affects their high-frequency dynamics. In order to understand its main features, we analytically studied a simple topologically disordered model, where the particles oscillate around randomly distributed centers, interacting through a generic pair potential. We present results of a resummation of the perturbative expansion in the inverse particle density for the dynamic structure factor and density of states. This gives accurate results for the range of densities found in real systems.Comment: Completely rewritten version, accepted in Physical Review Letter

Nonequilibrium functional RG with frequency dependent vertex function: A study of the single impurity Anderson model

We investigate nonequilibrium properties of the single impurity Anderson model by means of the functional renormalization group (fRG) within Keldysh formalism. We present how the level broadening Gamma/2 can be used as flow parameter for the fRG. This choice preserves important aspects of the Fermi liquid behaviour that the model exhibits in case of particle-hole symmetry. An approximation scheme for the Keldysh fRG is developed which accounts for the frequency dependence of the two-particle vertex in a way similar but not equivalent to a recently published approximation to the equilibrium Matsubara fRG. Our method turns out to be a flexible tool for the study of weak to intermediate on-site interactions U <= 3 Gamma. In equilibrium we find excellent agreement with NRG results for the linear conductance at finite gate voltage, magnetic field, and temperature. In nonequilibrium, our results for the current agree well with TD-DMRG. For the nonlinear conductance as function of the bias voltage, we propose reliable results at finite magnetic field and finite temperature. Furthermore, we demonstrate the exponentially small scale of the Kondo temperature to appear in the second order derivative of the self-energy. We show that the approximation is, however, not able to reproduce the scaling of the effective mass at large interactions.Comment: [v2] - minor changes throughout the text; added new Fig. 3; corrected pert.-theory data in Figs. 10, 11; published versio

Dynamic structure factor of the Ising model with purely relaxational dynamics

We compute the dynamic structure factor for the Ising model with a purely relaxational dynamics (model A). We perform a perturbative calculation in the $\epsilon$ expansion, at two loops in the high-temperature phase and at one loop in the temperature magnetic-field plane, and a Monte Carlo simulation in the high-temperature phase. We find that the dynamic structure factor is very well approximated by its mean-field Gaussian form up to moderately large values of the frequency $\omega$ and momentum $k$. In the region we can investigate, $k\xi \lesssim 5$, $\omega \tau \lesssim 10$, where $\xi$ is the correlation length and $\tau$ the zero-momentum autocorrelation time, deviations are at most of a few percent.Comment: 21 pages, 3 figure

Pushing the mass limit for intact launch and photoionization of large neutral biopolymers

Since their first discovery by Louis Dunoyer and Otto Stern, molecular beams have conquered research and technology. However, it has remained an outstanding challenge to isolate and photoionize beams of massive neutral polypeptides. Here we show that femtosecond desorption from a matrix-free sample in high vacuum can produce biomolecular beams at least 25 times more efficiently than nanosecond techniques. While it has also been difficult to photoionize large biomolecules, we find that tailored structures with an abundant exposure of tryptophan residues at their surface can be ionized by vacuum ultraviolet light. The combination of these desorption and ionization techniques allows us to observe molecular beams of neutral polypeptides with a mass exceeding 20,000 amu. They are composed of 50 amino acids – 25 tryptophan and 25 lysine residues – and 26 fluorinated alkyl chains. The tools presented here offer a basis for the preparation, control and detection of polypeptide beams

A new test for random number generators: Schwinger-Dyson equations for the Ising model

We use a set of Schwinger-Dyson equations for the Ising Model to check several random number generators. For the model in two and three dimensions, it is shown that the equations are sensitive tests of bias originated by the random numbers. The method is almost costless in computer time when added to any simulation.Comment: 6 pages, 3 figure

The search for magnetic fields in mercury-manganese stars

We performed a highly sensitive search for magnetic fields on a large set of HgMn stars. With the aid of a new polarimeter attached to the HARPS spectrometer at the ESO 3.6m-telescope, we obtained high-quality circular polarization spectra of 41 single and double HgMn stars. Using a multi-line analysis technique on each star, we co-added information from hundreds of spectral lines resulting in significantly greater sensitivity to the presence of magnetic fields, including very weak fields. For the 47 individual objects studied, including 6 components of SB2 systems, we do not detect any magnetic fields at greater than the 3 sigma level. The lack of detection in the circular polarization profiles indicates that if strong fields are present on these stars, they must have complex surface topologies. For simple global fields, our detection limits imply upper limits to the fields present of 2-10 Gauss in the best cases. We conclude that HgMn stars lack large-scale magnetic fields, typical for spotted magnetic Ap stars, sufficient to form and sustain the chemical spots observed on HgMn stars. Our study confirms that in addition to magnetically altered atomic diffusion, there exists another differentiation mechanism operating in the atmospheres of late-B main sequence stars which can compositional inhomogeneities on their surfaces.Comment: 12 pages, 8 figures, 2 table